WO2021110731A1 - Procédé pour déterminer la réponse de patients atteints d'un cancer de la prostate à un traitement avec des antagonistes du récepteur des androgènes sur la base de changements d'expression génique ou de profils de liaison à une protéine super-stimulatrice - Google Patents

Procédé pour déterminer la réponse de patients atteints d'un cancer de la prostate à un traitement avec des antagonistes du récepteur des androgènes sur la base de changements d'expression génique ou de profils de liaison à une protéine super-stimulatrice Download PDF

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WO2021110731A1
WO2021110731A1 PCT/EP2020/084239 EP2020084239W WO2021110731A1 WO 2021110731 A1 WO2021110731 A1 WO 2021110731A1 EP 2020084239 W EP2020084239 W EP 2020084239W WO 2021110731 A1 WO2021110731 A1 WO 2021110731A1
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treatment
patient
prostate cancer
antagonist
med1
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Simon BAUMGART
Ekaterina NEVEDOMSKAYA
Ralf Lesche
Bernard Haendler
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Bayer Aktiengesellschaft
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention refers to a method and kit for determining the response of prostate cancer patients to androgen receptor (AR) antagonists by determining the expression levels of selected genes or the protein binding and histone modification profiles at super-enhancers (SEs).
  • AR androgen receptor
  • SEs super-enhancers
  • the invention is related to kits to assess how a patient with prostate cancer responds to treatment with an AR antagonist, especially darolutamide.
  • the invention is related to the use of an AR antagonist, especially darolutamide, for the treatment of prostate cancer in a patient by analyzing a sample of body fluid or tumor tissue in vitro and determining how the patient responds to treatment.
  • AR stands for androgen receptor, NR3C4, AIS8, DHTR, HUMARA, HYSP1, KD, SBMA, SMAX1 and TFM.
  • AR gene In humans, it is encoded by the AR gene (Gene ID 367) and has the NCBI reference sequence identifier NM 000044.6. The corresponding AR protein has the identifier NP 000035.2.
  • Human AR cDNA and protein sequences are shown in J. Trapman et ak, Biochem. Biophys. Res. Commun., 1988, 153:241-248, C.S. Chang et ak, Science, 1988, 40:324-326 and D.B. Lubahn et ak, Science, 1988, 240:327-330.
  • AR is a nuclear receptor that is bound and activated by androgens, mainly dihydrotestosterone and testosterone. Upon ligand binding it translocates into the nucleus where it binds to specific DNA sequences and locally interacts with different cofactors, ultimately leading to stabilization and activation of the transcription complex and expression of downstream genes (B. Haendler, Biomed. Pharmacother., 2002, 56:78-83; F. Claessens et ak, Cell. Mol. Life Sck, 2017, 74:2217-2228).
  • the AR plays an essential role in early and late-stage prostate cancer (P.E. Lonergan and D.J. Tindall, J. Carcinog., 2011, 10:20; Q. Feng and B. He, Front Oneok, 2019, 9:858) and is therefore a target of choice.
  • MED1 stands for Mediator of RNA polymerase II transcription subunit 1. It is also named CRSP1, CRSP200, DRIP205, DRIP230, PBP, PPARBP, PPARGBP, RB18A, TRAP220 and TRIP2.
  • MED1 In humans it is encoded by the MED1 gene (Gene ID 5469) and has the sequence identifier NM 004774.4. The corresponding MED1 protein has the identifier NP 004765.2. Human MED1 cDNA and protein sequences are shown in P. Drane et ak, Oncogene, 1997, 15:3013-3024. MED1 belongs to the mediator complex, an essential coactivator for gene transcription by RNA polymerase II (M.T. Knuesel and D.J. Taatjes, Transcription, 2011, 2:28-31). MED1 interacts with the zinc finger transcription factor SP1 which binds to GC-rich regions often found in gene promoters (C. Rachez et al., Nature, 1999, 398:824-828). It binds to many other transcription factors, including the AR (J.W. Russo, Cancer Discov., 2019, 9:1490-1492).
  • FOXA1 stands for forkhead box protein Al. It is also named HNF3A, TCF3A.
  • FOXA1 In humans it is encoded by the FOXA1 gene (Gene ID 3169) and has the sequence identifier NM 004496.5. The corresponding FOXA1 protein has the identifier NP 004487.2.
  • Human FOXA1 cDNA and protein sequences are shown in E. Lai et al., Genes Dev., 1991, 5:416-427 and C.D. Bingle and S. Gowan, Biochim. Biophys. Acta, 1996, 1307:17-20 .
  • FOXA1 belongs to the forkhead family of DNA-binding proteins and acts as pioneer factor that opens up the chromatin structure and facilitates gene transcription (K.M. Jozwik and J.S. Carroll, Nat. Rev. Cancer, 2012, 12:381-385). It is an essential antagonist of the epithelial-to-mesenchymal cell transition (M. Katoh et al., Cancer Lett., 2013, 328: 198-206). It binds to the AR to modulate its function (Y. Zhao et al., Int. J. Biol. Sci., 2014, 10:614-619). FOXA1 mutations that define different prostate cancer subgroups have been identified (E.J. Adams et al., Nature, 2019, 57:408-412).
  • H3 stands for histone 3 family members. They are encoded by the following genes (Gene ID and NM identifier are given) which code for the proteins indicated under NP identifier :H3-3A (Gene ID: 3020; NM_002107.6; NP_002098.1), H3-3B (Gene ID: 3021; NM_005324.5; NP_005315.1), H3-4 (Gene ID: 8290; NM_003493.2; NP_003484.1), H3-5 (Gene ID: 440093; NM_001013699.3; NP_001013721.2), H3C1 (Gene ID 8350; NM_003529.2; NP_003529), H3C2 (Gene ID: 8358; NM_003537.3; NP_003528.1), H3C3 (Gene ID: 8352; NM_003531.2; NP_003522.1), H3C4 (Gene ID
  • Human histone H3s are encoded by several genes.
  • the main groups are H3.1, which includes HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I and HIST1H3J, H3.2, which includes HIST2H3A, HIST2H3C, HIST2H3D, and H3.3, which includes H3F3A and H3F3B.
  • H3.1 which includes HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I and HIST1H3J
  • H3.2 which includes HIST2H3A, HIST2H3C, HIST2H3D
  • H3.3 which includes H3F3A and H3F
  • Histone H3 proteins are part of globular nucleosomes and play an essential role in controlling access to DNA. They exist in several variants with highly conserved sequences. They undergo many post- translational modifications, especially acetylation, which is associated with active genes and accessible DNA regions. Lysine 27 (K27) is a main site of acetylation, but additional lysine residues such as K9, K14, K18 and K23 can also be acetylated in histone H3 (S. K. Kurdistani et al., Cell, 117:721-733).
  • SEs are dense cluster of genomic regions regulating gene transcription and which are dysregulated in various cancer types (Y. He et al., Frontiers Pharmacol., 2019, 10:361). These regions are highly bound by cell type-specific transcription factors and transcription-associated factors such as MED1, and are also highly enriched in histone acetylation, especially histone H3 acetylation (J. Loven et al., Cell, 2013, 153:320-334; W. A. Whyte et al., Cell, 2013, 153:307-319). SEs may represent specific vulnerabilities in different tumor types, including prostate cancer (V. Zuber et al., BMC Genomics, 2017, 18:270).
  • Targeted cancer drugs have a direct or indirect effect on one or more relevant biochemical pathways.
  • the serum levels of kallikrein-3/prostate-specific antigen are currently being used for prostate cancer screening and also for post-treatment monitoring and to provide guidance for subsequent therapies (B. Danielson et al., Can.
  • Identification of responders in the sense of the invention also means the identification of a patient or a group of patients with shared biological characteristics by using molecular, biochemical and diagnostic testing to select the optimal treatment for the patients and achieve the best possible outcome.
  • AR is used in the present invention for the AR gene (Gene ID 367, http://www.ncbi.nlm.nih.gov/gene/367) and AR protein (P10275.3).
  • MED1 is used in the present invention for the MED1 gene (Gene ID 5469, http://www.ncbi.nlm.nih.gov/gene/5469) and MED1 protein (AAH60758.1)
  • FOXA1 is used in the present invention for the FOXA1 gene (Gene ID 3169, http://www.ncbi.nlm.nih.gov/gene/3169) and FOXA1 protein (AAH33890.1).
  • SEs is used in the present invention for all genomic regions bound by the AR and identified using the ROSE algorithm and MED1 signals (Fig. 1).
  • the identified SE list is given in Table 4.
  • the invention is an in vitro analysis method for determining how a patient suffering from prostate cancer responds to treatment with an AR antagonist by: i) determining the expression levels of the genes described in Tables 1-3 by measurement of the respective mRNA or derived cDNA expression levels in a sample of body fluid or tumor cells or tumor tissue of a treated patient, and comparing them with those measured before treatment, or with healthy prostate tissue samples and/or ii) determining the binding levels of MED1, AR or FOXA1 or the histone acetylation levels, more specifically histone H3, more specifically histone H3 K27 acetylation, at the SEs listed in Table 4 in a sample of body fluid or tumor cells or tumor tissue of a treated patient, and comparing them with those measured before treatment, or with healthy prostate tissue samples, and wherein the presence in said in vitro sample of an altered mRNA or derived cDNA level and/or reduced MED1, AR or FOXA1 binding, or histone H3 acetylation levels, more specifically H3 K27 ace
  • the invention is an in vitro analysis method for determining how a patient suffering from prostate cancer responds to treatment with darolutamide by: i) determining the expression level of the response markers described in Tables 1-3 by measurement of the respective mRNA or derived cDNA expression levels in a sample of body fluid or tumor cells or tumor tissue of treated patient, and comparing them with those measured before treatment, and/or ii) determining the binding levels of MED1, AR or FOXA1 or the histone H3 acetylation levels, more specifically H3 K27 acetylation, at the SEs listed in Table 4 in a sample of body fluid or tumor cells or tumor tissue of a treated patient, and comparing them with those measured before treatment, and wherein the presence in said in vitro sample of a modified mRNA or derived cDNA level and/or reduced MED1, AR or FOX1 binding, or histone H3 acetylation levels, more specifically H3 K27 acetylation, at SEs following treatment with darolutamide in comparison with
  • the present invention concerns an analysis kit for monitoring the impact of an AR antagonist on treated prostate cancer patients.
  • Body fluid in the present invention means for example blood, plasma, serum, lymph, saliva, sweat, teardrops, urine or feces of a patient.
  • Tumor tissue in the present invention means for example primary tumor, metastases or circulating tumor cells.
  • RNA level in a sample is suggestive of a better response to the treatment of prostate cancer in the patient, if the mRNA, cDNA or protein expression level is at least 4-fold different than before treatment.
  • an expression level that is of at least 5 -fold different. It is also possible that an expression level is more than 6-fold different.
  • a reduced level of bound MED1 protein in a sample is suggestive of a better response to the treatment of prostate cancer in the patient, if the bound protein level is at least reduced 1.5 -fold than before treatment.
  • a bound protein level that is at least 2-fold lower. It is also possible that a bound protein level is more than 2-fold lower.
  • a reduced level of bound AR protein in a sample is suggestive of a better response to the treatment of prostate cancer in the patient, if the bound protein level is at least reduced 1.5 -fold than before treatment.
  • a bound protein level that is at least 2-fold lower. It is also possible that a bound protein level is more than 2-fold lower.
  • a reduced level of bound FOXA1 protein in a sample is suggestive of a better response to the treatment of prostate cancer in the patient, if the bound protein level is at least reduced 1.5 -fold than before treatment. More preferred is a bound protein level that is at least 2-fold lower. It is also possible that a bound protein level is more than 2-fold lower.
  • a reduced level of histone H3 acetylation levels, more specifically H3 K27 acetylation, in a sample is suggestive of a better response to the treatment of prostate cancer in the patient, if the bound protein level is at least reduced 1.5 -fold than before treatment.
  • histone H3 acetylation levels More preferred is a histone H3 acetylation levels, more specifically H3 K27 acetylation, that is of at least 2 -fold lower. It is also possible that the histone H acetylation levels, more specifically H3 K27 acetylation, is more than 2-fold lower
  • a further aspect of the invention is the use of the method for in vitro analysis of prostate cancer in a patient.
  • the patient is a mammal, especially a human.
  • RNA expression levels are assessed by determining the amount of RNA, for example mRNA or derived cDNA that is transcribed from a gene or gene sequence and coding for a peptide or protein.
  • Methods for gene expression analysis include, but are not limited to, reverse transcription quantitative PCR, differential display PCR, hybridization-based microarrays and next-generation sequencing, including RNA-Seq (F. Ozsolak and P. M. Milos, Nat. Rev. Genet. 2011, 12:87-98).
  • RNA For the measurement of gene expression, it is an advantage to amplify RNA, respectively cDNA.
  • Gene expression profiles indicative of AR antagonist responders are preferably those which show at least a 4-fold difference following AR antagonist treatment with regard to the expression of the respective mRNA or derived cDNA of the genes listed in Tables 1-3.
  • An expression difference of 4-fold is clearly predictive of the influence of the AR antagonist on the prostate cancer patient. More preferred is a difference of 5 -fold and much more preferred is a difference of 6-fold, which more clearly indicates that the AR antagonist will inhibit the progression of the tumor.
  • Protein extracts can be prepared by methods including, but not limited to, ion exchange column, size exclusion chromatography, SDS polyacrylamide gel electrophoresis, high performance liquid chromatography or reversed-phase chromatography (N. E. Labrou, Methods Mol. Biol., 2014, 1129:3-10).
  • Protein levels and protein occupancy at chromatin can be measured by methods including, but not limited to, protein immuno staining and microscopy, immunoprecipitation, Immunoelectrophoresis, Western blot, spectrophotometry, mass spectrometry, radioimmunoassay and enzyme-linked immunosorbent assay, immuno-PCR, stable isotope labeling by amino acids, tissue microarrays, protein biochips, chromatin immunoprecipitation (ChIP), ChIP with subsequent deep DNA sequencing (ChIP-seq), proteomics and nanoproteomics (K. K. Jain, J. BUON, 2007, Suppl. 1 : S31 -S38: A. Brewis and P. Brennan, Adv. Protein Chem. Struct.
  • Histone acetylation levels can be determined by liquid chromatography/mass spectrometry (Y. Zheng et al., Curr. Opin. Chem. Biol., 2016, 33:142-150), colorimetric and fluorometric assays, ELISA, Western blot analysis, immunocytochemistry, immunohistochemistry, ChIP, ChIP-seq (R.S. Jayani et al., Methods Cell Biol., 2010, 98:35-56), interaction assays based on fluorescence resonance energy transfer (K. Sasaki and M. Yoshida, Drug Discov. Today Technok, 2016, 19:51-56) or bioluminescence resonance energy transfer (M. Moustakim et al., Angew. Chem. Int. Ed. Engl., 2017, 56:827-831).
  • Protein occupancy and histone acetylation levels at SEs were measured by ChIP-seq in VCaP cells.
  • prostate cancer is understood as a disease of mammals, especially as a disease of the human and non-human mammal body, more specifically of the human body.
  • Prostate cancer in this regard means prostate adenocarcinoma, prostate glandular carcinoma, prostatic adenocarcinoma, prostate glandular cancer.
  • AR antagonists that are known are for example those compounds that are disclosed in E.D. Crawford and al (J. Urol., 2018, 200:956-966) and A.E. Dellis and A.G. Papatsoris (Expert Opin. Pharmacother., 2019, 20:163-172). They include competitive AR antagonists such as cyproterone acetate, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, darolutamide, proxalutamide.
  • body fluid or body tissue preferably blood, alternatively whole blood, serum or available plasma
  • body fluid or body tissue is taken from the patient to be examined, and the analysis is made in vitro, respectively ex vivo, which means outside the human body.
  • AR is to be understood as a human protein or polypeptide encoded by the AR gene shown in Gene ID 367 and having the amino acid sequence shown in P10275.3 , or a fragment of the AR protein sequence of at least 15 amino acids.
  • MED1 is to be understood as a human protein or polypeptide encoded by the MED1 gene shown in Gene ID 5469 and having the amino acid sequence shown in AAH60758.1, or a fragment of the MED1 protein sequence of at least 15 amino acids.
  • FOXA1 is to be understood as a human protein or polypeptide encoded by the FOXA1 gene shown in Gene ID 3169 and having the amino acid sequence shown in AAH33890.1, or a fragment of the FOXA1 protein sequence of at least 15 amino acids.
  • AR antagonists for example with one or more compounds selected from:
  • 1311-chTNT 1311-chTNT, abarelix, abiraterone, aclarubicin, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, Alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide,
  • the medication may comprise an AR antagonist and a therapeutic composition selected from the group consisting of tyrosine kinase inhibitors, CDK inhibitors, MEK inhibitors, PI3K inhibitors, MAP kinase inhibitors, Aik inhibitors, mTOR inhibitors, P-TEFb inhibitors, ATR inhibitors, PARP inhibitors, apoptosis modulators, hedgehog inhibitors, proteasome inhibitors, HDAC inhibitors, methotrexate, dexamethasone, PSMA-based compounds and combinations thereof.
  • a therapeutic composition selected from the group consisting of tyrosine kinase inhibitors, CDK inhibitors, MEK inhibitors, PI3K inhibitors, MAP kinase inhibitors, Aik inhibitors, mTOR inhibitors, P-TEFb inhibitors, ATR inhibitors, PARP inhibitors, apoptosis modulators, hedgehog inhibitors, proteasome inhibitors, HDAC inhibitors, methotrexate, dexamet
  • the invention further relates to a kit for in vitro analysis of the response of a prostate cancer patient to treatment with an AR antagonist containing the steps i) determining the expression level of the genes listed in Tables 1-3 by measurement of the respective mRNA or derived cDNA expression levels in a sample of body fluid or tumor tissue of a treated patient, and comparing the expression level with that before treatment, and/or ii) determining the bound MED1, AR or FOXA1, or histone H3 acetylation levels, more specifically H3 K27 acetylation, at the SEs listed in Table 4 in a sample of body fluid or tumor tissue of a treated patient, and comparing it with the levels before treatment, wherein the presence in said in vitro sample of a modified mRNA or derived cDNA and/or reduced bound MED1, AR or FOXA1, or reduced histone H3 acetylation levels, more specifically H3 K27 acetylation, following treatment with an AR antagonist in comparison with the untreated patient is suggestive of a better response
  • the determination of the expression level of the mRNA or derived cDNA and the determination of the bound MED1, AR or FOXA1, or histone H3 acetylation levels, more specifically H3 K27 acetylation can either be done combined, or separately.
  • all combinations are possible to get a valuable result for pharmacodynamic response.
  • ChIP Chromatin immunoprecipitation ChIP-seq Chromatin immunoprecipitation with deep sequencing chr Chromosome
  • Table 1 shows the genes with over 6-fold down or up-regulation following treatment of VCaP cells stimulated with 1 nM R1881 and the AR antagonist darolutamide (2 mM for 8 or 22 hours), in comparison to R1881 treatment only, as measured by sequencing on a hiSeq2500 device and mapping of FASTQ reads to the human genome GRCh38 and quantification with featureCounts from the Subread package. Differentially expressed genes were identified with DESeq2.
  • Table 2 shows the genes with 5- to 6-fold down or up-regulation following treatment of VCaP cells stimulated with 1 nM R1881 and the AR antagonist darolutamide (2 mM for 8 or 22 hours), in comparison to R1881 treatment only, as measured by sequencing on a hiSeq2500 device and mapping of FASTQ reads to the human genome GRCh38 and quantification with featureCounts from the Subread package. Differentially expressed genes were identified with DESeq2.
  • Table 3 shows the genes with 4- to 5-fold down or up-regulation following treatment of VCaP cells stimulated with 1 nM R1881 and the AR antagonist darolutamide (2 mM for 8 or 22 hours), in comparison to R1881 treatment only, as measured by sequencing on a hiSeq2500 device and mapping of FASTQ reads to the human genome GRCh38 and quantification with featureCounts from the Subread package. Differentially expressed genes were identified with DESeq2.
  • Table 4 shows the list of genomic coordinates (assembly GRCh37) of SEs changing in bound MED1,
  • Table 5 shows the ratio of medians of mean values of protein occupancy and H3K27 acetylation levels for the treatment conditions indicated, as depicted in Fig. 2.
  • Fig. 1 shows the detection of SEs at AR-binding genomic regions using the ROSE algorithm and MED1 signals as cut-off threshold.
  • Fig. 2 shows the averaged signals of transcription-associated factors and histone H3 K27 acetylation at scaled SE regions after treatment of VCaP cells with DMSO, R1881 (1 nM) or darolutamide (2 mM) + R1881 (1 nM).
  • Average signals for MED1 binding at SEs are reduced 2-fold between the R1881 and the R1881 + darolutamide groups.
  • Average signals for AR binding at SEs are reduced 1.5-fold between the R1881 and the R1881 + darolutamide groups.
  • Average signals for FOXA1 binding are reduced 1.5-fold between the R1881 and the R1881 + darolutamide groups.
  • Average signals for H3 K27 acetylation are reduced 1.5-fold between the R1881 and the R1881 + darolutamide groups.
  • BIOLOGICAL EXAMPLES The following examples describe the feasibility of the present invention.
  • the VCaP cell line (ATCC CRL2876) was selected as model for androgen-dependent prostate cancer.
  • VCaP cells were routinely cultured in an incubator at 37°C with 5% carbon dioxide in DMEM/glutamine medium.
  • the cells were starved for 2 days in RPMI1640 without phenol red supplemented with 10% charcoal-stripped, heat-inactivated and filtered fetal bovine serum, before treatment with the synthetic androgen R1881 (methyltrienolone) at a concentration of 1 nM.
  • darolutamide was added at a final concentration of 2 mM, and the cells were harvested after 8 or 22 hours post-treatment.
  • darolutamide was added at a concentration of 2 pM and the cells harvested 22 hours post-treatment.
  • RNA library preparation was performed after mRNA purification using poly-T beads, as described by the manufacturer (TruSeq Stranded mRNA Kit; Illumina, San Diego, CA, USA). Five biological replicates per condition were sequenced on a hiSeq2500 device via single-end, 50 base-pair reads with an average depth of 21 million reads per sample (Illumina, HiSeq2500 HTv4, SR, dual-indexing, 50 cycles).
  • FASTQ reads were mapped via STAR aligner to the human genome GRCh38 and quantified with featureCounts from the Subread package (Y. Liao, Nucleic Acids Res., 2019, 47:e47).
  • Differentially expressed genes were identified with DESeq2 (M.I. Love et al., Genome Biol., 2014, 15:550).
  • Significantly regulated genes were defined as having adjusted p-values lower than 0.05 and absolute log2- fold values higher than one for at least one time point.
  • ChIP experiments were performed in biological triplicates and library preparation was done as described by the manufacturer (MicroPlex Library Preparation Kit v2; Diagenode SA, Seraing, Belgium).
  • the libraries were sequenced on a HiSeq2500 Illumina machine with 50 base pair, single-end reads to an average depth of 25-30 million reads per sample.
  • the February 2009 human reference sequence (GRCh37) was used for mapping of the SE regions.
  • the ROSE algorithm was used to define SEs. Default conditions were applied, and transcriptional start sites regions excluded (J. Loven et al., Cell, 2013, 153:320-334; W.A. Whyte et al., Cell, 2013, 153:307- 319).
  • the MED1 signal was used to select groups of AR-binding regions qualifying as SEs with a cut-off set by the ROSE algorithm at 2737.7 (Fig. 1).
  • Table 3 List of gene transcripts down- or up-regulated 4- to 5-fold in the 2 mM darolutamide + 1 nM R1881 samples, compared to the 1 nM R1881 samples, following treatment for 8 or 22 hours.
  • the cut-off for SE definition is shown by the dotted horizontal grey line. Each red dot indicates an AR binding cluster either above or below the SE threshold.

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Abstract

La présente invention concerne un procédé et un kit pour déterminer l'impact d'antagonistes AR chez des patients atteints d'un cancer de la prostate par détermination des changements des niveaux d'expression de gènes sélectionnés et/ou la liaison de MED1 et/ou AR et/ou de FOXA1 et/ou d'acétylation de lysine d'histone H3 à des régions SE. Ceci peut être mesuré in vitro dans un échantillon de fluide corporel ou de tissu tumoral obtenu à partir de patients atteints d'un cancer de la prostate.
PCT/EP2020/084239 2019-12-03 2020-12-02 Procédé pour déterminer la réponse de patients atteints d'un cancer de la prostate à un traitement avec des antagonistes du récepteur des androgènes sur la base de changements d'expression génique ou de profils de liaison à une protéine super-stimulatrice WO2021110731A1 (fr)

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EP19213212.4 2019-12-03

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WO2014018926A1 (fr) * 2012-07-27 2014-01-30 Aragon Pharmaceuticals, Inc. Méthodes et compositions pour déterminer la résistance à une thérapie du récepteur des androgènes
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